Polymer stabilization of chromogen solutions

ABSTRACT

Disclosed embodiments concern a composition comprising DAB chromogen, and/or derivative thereof, a stabilizer, and polymer capable of preventing or reducing DAB precipitation relative to a composition that does not comprise the polymer. Also disclosed herein is a method for using the disclosed composition and embodiments of a kit.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/744,504, filed Jan. 18, 2013, which claims the benefit of U.S.Provisional Application No. 61/589,754, filed Jan. 23, 2012, thespecifications of which are incorporated herein by reference in theirentirety.

FIELD

The present disclosure concerns embodiments of a composition, a kit, andembodiments of a method for using such compositions, useful for a tissuestaining procedure wherein the composition comprises a diaminobenzidine(DAB) chromogen (and/or derivatives thereof) and a polymer capable ofreducing or substantially eliminating chromogen precipitation.

BACKGROUND

Certain commercially available DAB chromogen solutions (e.g. OPTIVIEW®DAB solution, Ventana Medical Systems, Inc. Tucson Ariz.) are stabilizedagainst oxidation. Sodium metabisulfite, for example, has been used as astabilizing solution at concentrations that do not significantly inhibitDAB tissue staining. Without being limited to a particular theory ofoperation, this antioxidant is believed (based on elemental analysis) toform an insoluble DAB hydrogen sulfate salt. This DAB hydrogen sulfatesalt has low aqueous solubility and readily precipitates from solution.The proposed process is illustrated below.

Tetramethyl benzidine chromogens (TMB) are known to associate withdextran sulfate. Dextran sulfate can maintain TMB oxidized productssoluble under processes normally used to precipitate TMB chromogenproducts in solution and also facilitate the TMB deposition in westernblotting. [Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9085-9089: “Use ofnonisotopic M13 probes for genetic analysis—Application to HLV class IIloci”, U.S. Pat. No. 4,789,630—“Ionic compounds containing the cationicmeriquinone of a benzidine” and U.S. Pat. No. 5,013,646 orWO199100667—“TMB formulation for soluble and precipitable HRP-ELISA”].There still exists a need in the art, however, to address the problem ofDAB-sulfate precipitation prior to DAB oxidation in tissue stainingmethods.

SUMMARY

The present disclosure concerns a composition, a method for detecting atarget within a tissue sample with that composition, and a kit forchromogenic immunohistochemistry. The composition comprises a DABchromogen at a sufficient concentration and with sufficient stability tobe used as a chromogen for the detection of targets inimmunohistochemistry. Sufficient stability includes stability in storagesuch that after extended periods of time (e.g. 6 months, 12 months, 18months, and 24 months) the chromogen solution can still be used todetect targets in tissue samples without a meaningful decrease in signalcompared to the same chromogen solution freshly prepared. Sufficientstability also includes sufficient instability so that when used indetection, sufficient chromogen precipitates to detect the targets in atissue sample.

In illustrative embodiments, a composition for chromogenicimmunohistochemistry includes a solvent, a DAB chromogen, a stabilizer,and a polymer soluble in the solvent. In one embodiment, the polymerreduces DAB precipitation from the solution under accelerated agingconditions relative to a like solution not including the polymer. Inanother embodiment, the polymer comprises a sulfate functional group, asulfonate functional group, an amine functional groups, or combinationsthereof. In another embodiment, the amine functional groups include aprimary amine, a secondary amine, a tertiary amine, or a quaternaryamine. In yet another embodiment, the polymer is a polyalkyleneaminepolymer. In one embodiment, the polymer is selected from dextransulfate, polystyrene sulfonate, polyethyleneimine, aminodextran, dextranDEAE, polystyrene sulfonate maleic acid co-polymer, polyvinylsulfonate,poly(2-vinyl-1-methylpyridinium bromide,poly(2-methylacryloxethyltrimethylammonium bromide,poly(acrylamide/2-methylacryloxethyltrimethylammonium bromide, andcombinations thereof. In another embodiment, the polymer is a linear orbranched polyethyleneimine

In illustrative embodiments, a composition for chromogenicimmunohistochemistry comprises a DAB chromogen and a polymer having aformula selected from

wherein each Y independently is selected from oxygen, sulfur, and NR^(a)wherein R^(a) is selected from hydrogen, aliphatic, aryl, heteroaryl,and heteroaliphatic; R¹ is selected from aliphatic, aryl, heteroaryl, ora heteroatom-containing moiety, selected from an ester, acid, and amide;R² is selected from hydrogen, aliphatic, heteroaliphatic, aryl, orheteroaryl; R³ is a polymer functional group selected from a sulfonate,an amine, a carboxyl group, a carboxylate, or combinations thereof; andn ranges from 1 to about 100. In one embodiment, the polymer has aformula of

wherein R² is selected from hydrogen, aliphatic, heteroaliphatic, aryl,or heteroaryl; R³ is a polymer functional group selected from asulfonate, an amine, a carboxyl group, a carboxylate, or combinationsthereof; R⁴ is selected from hydrogen, aliphatic, and(CH₂)_(q)NR^(a)R^(b)R^(c) wherein R^(a), R^(b), and R^(c) independentlyare selected from hydrogen, aliphatic, aryl, and combinations thereof; qranges from 1 to about 10; and n ranges from 1 to about 100. In anotherembodiment, the polymer is a co-polymer comprising two or more monomers.

In illustrative embodiments, a composition for chromogenicimmunohistochemistry includes an enhancer selected from the groupconsisting of imidazole, 2-hydroxypyridine, and combinations thereof. Inanother embodiment, the composition includes the stabilizer sodiummetabisulfite. In one embodiment, the polymer reduces DAB precipitationby about 10% to about 100% under accelerated aging conditions relativeto the composition that does not include the polymer, determined byeither HPLC in an accelerated aging study at 45° C. or by polymerscreening using KHSO₄. In another embodiment, the polymer reduces DABprecipitation under accelerated aging conditions relative to thecomposition that does not include the polymer while precipitatingequivalently to the composition that does not include the polymer underdetection conditions. Illustratively, precipitating equivalentlyincludes generating no statistically significant deviation in averageobserved staining intensity, as determined by a qualified reader,compared to the composition that does not include the polymer. Furtherillustratively, the polymer is configured to form a complex with the DABchromogen so as to maintain solubility of the DAB chromogen in thesolvent. In one embodiment, the stabilizer and the polymer substantiallyprevent the DAB chromogen from decomposing and/or being oxidized understorage conditions.

In illustrative embodiments, a composition for chromogenicimmunohistochemistry detection includes a DAB chromogen, an enhancer, astabilizer, and a polymer; wherein the enhancer is imidazole or2-hydroxypyridine, the stabilizer is sodium metabisulfite or sodiumbisulfite, and the polymer is dextran sulfate, polystyrene sulfonate,polyethyleneimine, aminodextran, dextran DEAE, polystyrene sulfonatemaleic acid co-polymer, polyvinylsulfonate,poly(2-vinyl-1-methylpyridinium bromide,poly(2-methylacryloxethyltrimethylammonium bromide, orpoly(acrylamide/2-methylacryloxethyltrimethylammonium bromide; whereinthe detection solution is configured to remain stable under storageconditions so that the polymer complexes the DAB chromogen or a productthereof so as to maintain the amount of the DAB chromogens in thesolution; wherein the detection solution is configured so that the DABprecipitates under detection conditions produces a signal suitable forchromogenic immunohistochemistry. In one embodiment, the DAB chromogenis 3,3′-diaminobenzidine, the stabilizer is sodium metabisulfite, andthe polymer is polyethyleneimine. In another embodiment, the compositioncomprises about 1 to about 15 mM 3,3′-diaminobenzidine, about 1 to about20 mM imidazole, about 1 to about 20 mM 2-hydroxypyridine, about 0.1 toabout 3 mM sodium metabisulfite and about 0.05% to about 0.5% (w/w)polyethyleneimine.

A method for detecting a target in a tissue sample with a DAB chromogenincludes contacting the tissue sample with a specific binding moiety,the specific binding moiety being specific to the target, labeling thespecific binding moiety with an enzyme conjugate, contacting the tissuesample with a chromogen solution, the chromogen solution comprising DABor a derivative thereof, a stabilizer, and a polymer configured toreduce DAB precipitation under storage conditions but maintainprecipitation under detection conditions, contacting the tissue samplewith an oxidant, wherein a reaction between the oxidant, the chromogensolution, and the enzyme conjugate causes the DAB to deposit proximallyto the target, contacting the tissue sample with a counterstain, anddetecting the target in the tissue sample by locating the DAB chromogen.In one embodiment, the tissue sample is a formalin-fixed,paraffin-embedded tissue sample. In another embodiment, storageconditions include a sealed storage container not in fluid communicationwith other reagents and detection conditions include deposited on thetissue sample and in contact with the enzyme. In another embodiment, thespecific binding moiety is an antibody or a nucleic acid probe. In yetanother embodiment, labeling the specific binding moiety includesdetecting a hapten selected from an oxazole, a pyrazole, a thiazole, anitroaryl compound other than dinitrophenyl, a benzofurazan, atriterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, aheterobiaryl, an azo aryl, or a benzodiazepine with an enzyme conjugateincluding an anti-hapten antibody specific to the hapten. In variousembodiments, the hapten is selected from HQ, DIG, DNP, TS, NP, DCC, andbiotin. In one embodiment, the enzyme is a peroxidase. For example, theenzyme is horseradish peroxidase. In yet other embodiments, contactingthe tissue sample with a chromogen solution comprises adding a firstcomponent solution comprising the DAB or derivative thereof, a secondcomponent solution comprising the stabilizer, and a third componentsolution comprising the polymer to the tissue sample to form thechromogen solution while in contact with the tissue sample. In furtherembodiments, the method includes contacting the tissue sample with a DABcomposition as described herein.

In illustrative embodiments, a method of detecting a target in aformalin-fixed, paraffin-embedded tissue sample includes contacting thetissue sample with a haptinylated antibody, contacting the tissue samplewith an enzyme conjugate comprising an anti-hapten antibody andhorseradish peroxidase, contacting the tissue sample with a chromogencomposition, the chromogen composition comprising a DAB chromogen, orderivative thereof, an enhancer selected from imidazole,2-hydroxypyridine, and combinations thereof; sodium metabisulfite; and apolymer selected from dextran sulfate, polystyrene sulfonate,polyethyleneimine, aminodextran, dextran DEAE, polystyrene sulfonatemaleic acid co-polymer, polyvinylsulfonate,poly(2-vinyl-1-methylpyridinium bromide,poly(2-methylacryloxethyltrimethylammonium bromide,poly(acrylamide/2-methylacryloxethyltrimethylammonium bromide, andcombinations thereof; contacting the tissue sample with hydrogenperoxidase; contacting the tissue sample with a hematoxylincounterstain, and detecting the target in the tissue sample. In furtherillustrative embodiments, a kit for chromogenic detection of tissuesamples includes a DAB chromogen, an enhancer, a stabilizer, and apolymer capable of reducing or preventing DAB precipitation relative toa composition that does not include the polymer, the DAB chromogen andthe polymer packaged in a first dispenser or bottle. In one embodiment,the enhancer is selected from imidazole, 2-hydroxypyridine, andcombinations thereof and the stabilizer is sodium metabisulfite. Inanother embodiment, the kit further includes an enzyme and antibodyconjugate packaged in a second dispenser or bottle. In anotherembodiment, the polymer provides from greater than 10% to about 50%reduction in precipitation as determined by either HPLC in anaccelerated aging study at 45° C. or by polymer screening using KHSO₄.In another embodiment, the antibody is an anti-hapten antibody and theenzyme is a horseradish peroxidase. In another embodiment, the kitfurther includes hydrogen peroxide packaged in a third dispenser orbottle. In one embodiment, the polymer is present at a concentrationeffective to reduce precipitation of the DAB chromogen in the firstdispenser or bottle while the concentration is also effective forallowing precipitation upon contacting the DAB chromogen with the enzymeand antibody conjugate and the hydrogen peroxide. Illustratively,allowing precipitation includes generating no statistically significantdeviation in average observed staining intensity as determined by aqualified reader compared to a comparative chromogenic detectioncomposition that does not include the polymer.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image of a control slide wherein the tonsil tissue sampleis stained for Ki67 with a DAB chromogen solution.

FIG. 2 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and dextran sulfate (Mw=9-20 kDa, 2 wt %).

FIG. 3 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution.

FIG. 4 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and polystyrene sulfonate (PSS) polymer having aMw=1.5 kDa (5 wt %).

FIG. 5 is an image of a tonsil tissue section stained with for Ki67 aDAB chromogen solution and PSS polymer having a Mw=5.1 kDa (5 wt %).

FIG. 6 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and PSS polymer having a Mw=7.5 kDa (5 wt %).

FIG. 7 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and PSS polymer having a Mw=16 kDa (5 wt %).

FIG. 8 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and PSS polymer having a Mw=35 kDa (5 wt %).

FIG. 9 is an image of a control slide wherein the tonsil tissue sampleis stained for Ki67 with a DAB chromogen solution.

FIG. 10 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and aminodextran polymer having a Mw=10 kDa.

FIG. 11 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and aminodextran polymer having a Mw=40 kDa.

FIG. 12 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and aminodextran polymer having a Mw=70 kDa.

FIG. 13 is an image of a representative HPLC trace and a spectrum indexplot.

FIG. 14 is an image of a control slide wherein the tonsil tissue sampleis stained for Ki67 with a DAB chromogen solution.

FIG. 15 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and 4 wt % polystyrene sulfonate (Mw=1.5 kDa).

FIG. 16 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and 4 wt % dextran sulfate (Mw=6.5-10 kDa).

FIG. 17 is an image of a control slide wherein the tonsil tissue sampleis stained for Ki67 with a DAB chromogen solution.

FIG. 18 is an image of a tonsil tissue section stained for Ki67 with aDAB chromogen solution and 5 wt % polyallylamine (Mw=15 kDa).

FIG. 19 is an image of a tonsil tissue sample stained for Ki67 with asolution of a DAB chromogen and 0.1 wt % of linear polyethyleneimine(Mw=2.5 kDa).

FIG. 20 is an image of a tonsil tissue sample stained for Ki67 with asolution of a DAB chromogen and 0.5 wt % of linear polyethyleneimine(Mw=2.5 kDa).

FIG. 21 is an image of a tonsil tissue sample stained for Ki67 with asolution of a DAB chromogen and 1.0 wt % of linear polyethyleneimine(Mw=2.5 kDa).

FIG. 22 is an image of a tonsil tissue sample stained for Ki67 with asolution of a DAB chromogen and 3.0 wt % of linear polyethyleneimine(Mw=2.5 kDa).

FIG. 23 is an image of a control slide wherein the tonsil tissue sampleis stained for Ki67 with a solution of a DAB chromogen.

FIG. 24 is an image of a tonsil tissue sample stained for Ki67 with asolution of a DAB chromogen and 7 wt % of dextran DEAE (Mw=500 kDa).

FIG. 25 is an image of a tonsil tissue sample stained for Ki67 with asolution of a DAB chromogen and 2 wt % of linear polyethyleneimine(Mw=2.5 kDa).

DETAILED DESCRIPTION I. Introduction

The present disclosure concerns a composition comprising a DABchromogen, and/or derivatives thereof, and a polymer that includes oneor more functional groups capable of forming a complex with thechromogen, the counter-ion salt, or the sulfate product of bisulfiteoxidation during formulation. An amount of the polymer is used tosufficiently reduce or substantially prevent precipitation of thechromogen. The disclosed composition may be used to increase or improvechromogen deposition during a staining procedure, such as tissuestaining. Also disclosed is a method of using the disclosed compositionand kits comprising the disclosed composition.

II. Definitions and Abbreviations

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Publishers, 1994); Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by Wiley, John & Sons, Inc., 1995; and George P.Rédei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics,2nd Edition, 2003.

The singular forms “a,” “an,” and “the” refer to one or more than one,unless the context clearly dictates otherwise. For example, the term“comprising a cell” includes single or plural cells and is consideredequivalent to the phrase “comprising at least one cell.” The term “or”refers to a single element of stated alternative elements or acombination of two or more elements, unless the context clearlyindicates otherwise. A wavy line (“

”), is used to indicate a bond disconnection, and a dashed line (“- --”) is used to illustrate that a bond may be formed at a particularposition.

Although methods and materials similar or equivalent to those describedherein can be used to practice or test the disclosed technology,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art to practice the present disclosure.

Analog, Derivative or Mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound.

Aryl: A substantially hydrocarbon-based aromatic compound, or a radicalthereof (e.g. C₆H₅) as a substituent bonded to another group,particularly other organic groups, having a ring structure asexemplified by benzene, naphthalene, phenanthrene, anthracene, etc. Thisterm also encompasses substituted aryl compounds.

Avidin: Any type of protein that specifically binds biotin to thesubstantial exclusion of other small molecules that might be present ina biological sample. Examples of avidin include avidins that arenaturally present in egg white, oilseed protein (e.g., soybean meal),and grain (e.g., corn/maize) and streptavidin, which is a protein ofbacterial origin.

Branched: This term refers to a compound and/or functional groupcomprising one or more units comprising a carbon atom that is bound toat least three other carbon atoms.

Chromogen: A substance capable of conversion to and/or deposition of acolored product, such as a pigment or dye. Certain chromogens areelectron donors that, when oxidized, become a colored product.Production of a colored product, and/or the property of becominginsoluble upon chemical conversion, such as by oxidation, makechromogens useful for IHC. Particular examples of chromogenic compounds,without limitation, include 3,3′-diaminobenzidine (DAB),tetramethylbenzidine (TMB), 2,2′-azino-di[3-ethylbenzothiazolinesulphonate] (ABTS), iodonitrotetrazolium (INT), tetrazolium blue andtetrazolium violet. DAB is a chromogen that produces a brown end product(e.g. through an enzymatic reaction, such as HRP) that is highlyinsoluble in aqueous solutions.

Conditions sufficient to detect: Any environment that permits thedesired activity, for example, that permits a probe to bind a target andthe interaction to be detected. For example, such conditions includeappropriate temperatures, buffer solutions, and detection means such asmicroscopes and digital imaging equipment.

Contacting: Placement that allows association between two or moremoieties, particularly direct physical association, for example both insolid form and/or in liquid form (for example, the placement of abiological sample, such as a biological sample affixed to a slide, incontact with a composition, such as a solution containing thecompositions disclosed herein).

Control: A sample or procedure performed to assess test validity. In oneexample, a control is a quality control, such as a positive control. Forexample, a positive control is a procedure or sample, such as a tissueor cell, that is similar to the actual test sample, but which is knownfrom previous experience to give a positive result. A positive controlconfirms that the basic conditions of the test produce a positiveresult, even if none of the actual test samples produce such result. Ina particular example, a positive control is a sample known by previoustesting to contain the suspected antigen. In other examples, a controlis a negative control. A negative control is a procedure or test sampleknown from previous experience to give a negative result. The negativecontrol demonstrates the base-line result obtained when a test does notproduce a measurable positive result; often the value of the negativecontrol is treated as a “background” value to be subtracted from thetest sample results. In a particular example, a negative control is areagent that does not include the specific primary antibody. Otherexamples include calibrator controls, which are samples that contain aknown amount of a control antigen. Such calibrator controls have anexpected signal intensity, and therefore can be used to correct forinter- or intra-run staining variability.

Detergent or Surfactant: A substance that reduces the surface tension ofwater. Specifically, a detergent or surfactant is a surface-active agentthat concentrates at oil-water interfaces and exerts an emulsifyingaction. Detergents are classified as anionic, cationic, or nonionic,depending on their mode of chemical action. Nonionic detergents functionvia a hydrogen-bonding mechanism. Further, surfactants or detergentsreduce interfacial tension between two liquids. A surfactant moleculetypically has a polar or ionic “head” and a nonpolar hydrocarbon “tail.”Upon dissolution in water, the surfactant molecules aggregate and formmicelles, in which the nonpolar tails are oriented inward and the polaror ionic heads are oriented outward toward the aqueous environment. Thenonpolar tails create a nonpolar “pocket” within the micelle. Nonpolarcompounds in the solution are sequestered in the pockets formed by thesurfactant molecules, thus allowing the nonpolar compounds to remainmixed within the aqueous solution.

Detect: To determine if an agent (such as a signal or particularantigen, protein or nucleic acid) is present or absent, for example, ina sample. In some examples, this can further include quantification,and/or localization, for example localization within a cell orparticular cellular compartment. “Detecting” refers to any method ofdetermining if something exists, or does not exist, such as determiningif a target molecule is present in a biological sample. For example,“detecting” can include using a visual or a mechanical device todetermine if a sample displays a specific characteristic. In certainexamples, detection refers to visually observing a probe bound to atarget, or observing that a probe does not bind to a target. Forexample, light microscopy and other microscopic means are commonly usedto detect chromogenic precipitates for methods described here.

Enhanc(e/er/ement/ing): An enhancer or enhancing reagent is any compoundor any combination of compounds sufficient to increase the catalyticactivity of an enzyme, as compared to the enzyme activity without suchcompound(s) Enhancer(s) or enhancing reagent(s) can also be defined as acompound or combination of compounds that increase or accelerate therate of binding an activated conjugate to a receptor site.Enhanc(e/ement/ing) is a process by which the catalytic activity of anenzyme is increased by an enhancer, as compared to a process that doesnot include such an enhancer Enhanc(e/ement/ing) can also be defined asincreasing or accelerating the rate of binding of an activated conjugateto a receptor site. Enhanc(e/ement/ing) can be measured visually, suchas by scoring by a pathologist. In particular embodiments, scores rangefrom greater than 0 to greater than 4, with the higher number indicatingbetter visual detection. More typically, scores range from greater than0 to about 4++, such as 1, 1.5, 2, 2.5, 3, 3.5, 3.75, 4, 4+, and 4++. Inaddition, enhanc(e/ement/ing) can be measured by determining theapparent V_(max) of an enzyme. In particular embodiments, the termencompasses apparent V_(max) values (measured as optical density/minute)ranging from greater than 0 mOD/min to about 400 mOD/min, such as about15 mOD/min, 18 mOD/min, about 20 mOD/min, about 40 mOD/min, about 60mOD/min, about 80 mOD/min, about 100 mOD/min, about 120 mOD/min, about140 mOD/min, about 160 mOD/min, about 200 mOD/min, about 250 mOD/min,about 300 mOD/min, about 350 mOD/min, and about 400 mOD/min. Moretypically, the V_(max) ranges from greater than 0 mOD/min to about 160mOD/min, such as about 20 mOD/min, about 40 mOD/min, about 60 mOD/min,about 80 mOD/min, about 100 mOD/min, about 120 mOD/min, about 140mOD/min, and about 160 mOD/min. In addition, enhancement can occur usingany concentration of an enhancer greater than 0 mM. Typically,enhancement occurs at enhancer concentrations ranging from great thanabout 0.01 mM to about 100 mM, such as about 0.01 mM, about 0.02 mM,about 0.05 mM, about 0.10 mM, about 0.20 mM, about 0.50 mM, about 1.0mM, about 2.0 mM, about 3.0 mM, about 5.0 mM, about 10.0 mM, about 20.0mM, about 30.0 mM, about 40.0 mM, about 50.0 mM, about 75.0 mM, or about100.0 mM, such as about 0.01 mM to about 0.10 mM, about 0.05 mM to about0.50 mM, about 0.4 mM to about 1.0 mM, about 0.5 mM to about 2.0 mM,about 1.0 mM to about 10.0 mM, about 5.0 mM to about 50.0 mM, and about20.0 mM to about 100.0 mM Enhanc(e/er/ement/ing), in general and speciesthereof are disclosed in assignee's co-pending application U.S.Publication No. 2012/0171668, which is incorporated herein by reference.

Fixation: A process which preserves cells and tissue constituents in asclose to a life-like state as possible and allows them to undergopreparative procedures without change. Fixation arrests the autolysisand bacterial decomposition processes that begin upon cell death, andstabilizes the cellular and tissue constituents so that they withstandthe subsequent stages of tissue processing, such as for IHC. Tissues maybe fixed by either perfusion with or submersion in a fixative, such asan aldehyde (such as formaldehyde, paraformaldehyde, glutaraldehyde, andthe like). Other fixatives include oxidizing agents (for example,metallic ions and complexes, such as osmium tetroxide and chromic acid),protein-denaturing agents (for example, acetic acid, methanol, andethanol), fixatives of unknown mechanism (for example, mercuricchloride, acetone, and picric acid), combination reagents (for example,Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman'sfluid, and Gendre's fluid), microwaves, and miscellaneous (for example,excluded volume fixation and vapour fixation). Additives also may beincluded in the fixative, such as buffers, detergents, tannic acid,phenol, metal salts (for example, zinc chloride, zinc sulfate, andlithium salts), and lanthanum. The most commonly used fixative inpreparing samples for IHC is formaldehyde, generally in the form of aformalin solution (4% formaldehyde in a buffer solution, referred to as10% buffered formalin).

Hapten: A molecule, typically a small molecule that can combinespecifically with an antibody, but typically is substantially incapableof being immunogenic except in combination with a carrier molecule.Exemplary haptens include an oxazole, a pyrazole, a thiazole, anitroaryl compound other than dinitrophenyl, a benzofurazan, atriterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, aheterobiaryl, an azo aryl, or a benzodiazepine. More particularly, thehapten may be 2,4-dinitrophenyl (DNP), biotin, digoxigenin,5-nitro-3-pyrazolecarbamide (NP), 3-hydroxy-2-quinoxalinecarbamide (HQ),2-acetamido-4-methyl-5-thiazolesulfonamide (TS), and4-(diethylamino)azobenene-4′-sulfonamide (DABSYL), or7-(diethylamino)-2-oxo-2H-chromene-3-carboxyylic acid (DCC).

Immunohistochemistry (IHC): A method of determining the presence ordistribution of an antigen in a sample by detecting interaction of theantigen with a specific binding agent or moiety, such as an antibody. Asample including an antigen (such as a target antigen) is incubated withan antibody under conditions permitting antibody-antigen binding.Antibody-antigen binding can be detected by means of a detectable labelconjugated to the antibody (direct detection) or by means of adetectable label conjugated to a secondary antibody, which is raisedagainst the primary antibody (e.g., indirect detection). Detectablelabels include, but are not limited to, radioactive isotopes,fluorochromes (such as fluorescein derivatives, and rhodaminederivatives), enzymes and chromogenic molecules.

In situ hybridization (ISH): A type of hybridization that uses a labeledcomplementary DNA or RNA strand (i.e., probe) to localize a specific DNAor RNA sequence in a portion or section of tissue (in situ), or, if thetissue is small enough (e.g., plant seeds, Drosophila embryos), in theentire tissue (whole mount ISH). This is distinct fromimmunohistochemistry, which localizes proteins in tissue sections. DNAISH can be used to determine the structure of chromosomes, such as foruse in medical diagnostics to assess chromosomal integrity. RNA ISH(hybridization histochemistry) is used to measure and localize mRNAs andother transcripts within tissue sections or whole mounts. Forhybridization histochemistry, sample cells and tissues are usuallytreated to fix the target transcripts in place and to increase access ofthe probe to the target molecule. As noted above, the probe is either alabeled complementary DNA or a complementary RNA (Riboprobe). The probehybridizes to the target sequence at elevated temperature, and then theexcess probe is washed away (optionally hydrolyzed using RNase in thecase of unhybridized, excess RNA probe). Solution parameters, such astemperature, salt and/or detergent concentration, can be manipulated toremove any non-identical interactions (i.e. only exact sequence matcheswill remain bound). Then, the labeled probe having been labeledeffectively, such as with either radio-, fluorescent- or antigen-labeledbases (e.g., digoxigenin), is localized and potentially quantified inthe tissue using either autoradiography, fluorescence microscopy orimmunohistochemistry, respectively.

Molecule of interest or Target: A molecule for which the presence,location and/or concentration is to be determined. Examples of moleculesof interest include proteins and nucleic acid sequences tagged withhaptens.

Multiplex, -ed, -ing: Embodiments of the present disclosure allowmultiple targets in a sample to be detected substantiallysimultaneously, or sequentially, as desired, using plural differentconjugates. Multiplexing can include identifying and/or quantifyingnucleic acids generally, DNA, RNA, peptides, proteins, both individuallyand in any and all combinations. Multiplexing also can include detectingtwo or more of a gene, a messenger and a protein in a cell in itsanatomic context.

Sample: The term “sample” refers to any liquid, semi-solid or solidsubstance (or material) in or on which a target can be present. Inparticular, a sample can be a biological sample or a sample obtainedfrom a biological material. Examples of biological samples includetissue samples and cytology samples. In some examples, the biologicalsample is obtained from an animal subject, such as a human subject. Abiological sample is any solid or fluid sample obtained from, excretedby or secreted by any living organism, including without limitation,single celled organisms, such as bacteria, yeast, protozoans, and amebasamong others, multicellular organisms (such as plants or animals,including samples from a healthy or apparently healthy human subject ora human patient affected by a condition or disease to be diagnosed orinvestigated, such as cancer). For example, a biological sample can be abiological fluid obtained from, for example, blood, plasma, serum,urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreoushumor, or any bodily secretion, a transudate, an exudate (for example,fluid obtained from an abscess or any other site of infection orinflammation), or fluid obtained from a joint (for example, a normaljoint or a joint affected by disease). A biological sample can also be asample obtained from any organ or tissue (including a biopsy or autopsyspecimen, such as a tumor biopsy) or can include a cell (whether aprimary cell or cultured cell) or medium conditioned by any cell, tissueor organ. In some examples, a biological sample is a nuclear extract. Insome examples, a biological sample is bacterial cytoplasm. In otherexamples, a sample is a test sample. For example, a test sample is acell, a tissue or cell pellet section prepared from a biological sampleobtained from a subject. In an example, the subject is one that is atrisk or has acquired a particular condition or disease.

Specific binding moiety: A member of a specific-binding pair. Specificbinding pairs are pairs of molecules that are characterized in that theybind each other to the substantial exclusion of binding to othermolecules (for example, specific binding pairs can have a bindingconstant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹greater than a binding constant for either of the two members of thebinding pair with other molecules in a biological sample). Particularexamples of specific binding moieties include specific binding proteins(for example, antibodies, lectins, avidins such as streptavidins, andprotein A), nucleic acids sequences, and protein-nucleic acids. Specificbinding moieties can also include the molecules (or portions thereof)that are specifically bound by such specific binding proteins.

Stabilizer: A compound capable of substantially preventing a chromogenfrom decomposition, precipitation, and/or oxidation. In one embodiment,a stabilizer is an antioxidant, such as sodium metabisulfite.

Tissue: A collection of interconnected cells that perform a similarfunction within an organism.

III. Chromogen-Polymer Composition

Certain disclosed embodiments concern a composition comprising achromogen, particularly a DAB chromogen, and/or derivatives thereof, anda polymer capable of forming a complex with the chromogen, thecounter-ion salt, or sulfate product of bisulfite oxidation. Thedisclosed composition may be used in colorimetric procedures, such astissue staining. A person of ordinary skill in the art will appreciatethat a composition useful for a particular protocol may vary from thatof a different protocol, and still be within the scope of thecomposition disclosed herein. Moreover, various optional materials, suchas having different reagent concentrations or ratios of reagentsamounts, may be included in useful compositions formulated according tothe present invention, including, but not limited to, stabilizers,enhancers, counterstains, and/or buffers.

The disclosed composition may be used to increase chromogen deposition,particularly DAB deposition, in staining procedures, such as tissuestaining. Table 1 details results obtained from particular workingembodiments wherein a base DAB chromogen solution (DAB, imidazole,2-hydroxypyridine, and sodium metabisulfite) containing a polymeraccording to the present disclosure was exposed to varyingconcentrations of potassium hydrogen sulfate (KHSO₄).

TABLE 1 Polymer screening system 5.5 μmol DAB + 0 μmol 1.37 μmol 2.75μmol 8.0 μmol 5 wt % Polymer KHSO₄ KHSO₄ KHSO₄ KHSO₄ No Polymer no pptppt ppt ppt 15-20 kDa Dextran no ppt no ppt ppt ppt 9-20 kDa DextranSulfate no ppt no ppt no ppt no ppt

Sodium metabisulfite has been used as an antioxidant stabilizer atconcentrations that do not significantly inhibit DAB tissue staining.Without being limited to a particular theory of operation, thisstabilizer forms an oxidized hydrogen sulfate byproduct which isbelieved to form insoluble DAB hydrogen sulfate salt precipitate (basedon elemental analysis). This DAB hydrogen sulfate salt has a low aqueoussolubility and readily precipitates from solution.

HPLC analysis was performed on the polymer-stabilized DAB chromogen tojudge heat stress stability (at 45° C.) and each polymer's ability toinhibit DAB sulfate precipitate before and after addition of potassiumhydrogen sulfate addition. Samples were analyzed for residual DABconcentration (analytical ultracentrifugation, “AUC”) relative to2-hydroxypyridine (AUC) as an internal standard. The 2-hydroxypyridineconcentration was shown to not be influenced by either heat stress orpotassium hydrogen sulfate addition. The DAB concentration (AUC)diminished if the DAB tetrahydrochloride formed an insoluble DABhydrogen sulfate precipitate. This precipitate can be removed from thesample using a pre-column HPLC filter and is, thus, not detectable byHPLC. Thermal stress was shown to expedite DAB oxidation. Thesebyproducts did not accumulate in solution to any substantialconcentration and precipitated from solution. Polymers that provided thegreatest DAB thermal stability and largest inhibition of DAB sulfateprecipitate formation became leading candidates.

As Table 1 indicates, DAB sulfate precipitation was prevented incompositions comprising the chromogen solution and particularembodiments of the disclosed polymer. FIGS. 1 and 2 are images thatillustrate the results obtained from particular working embodiments.FIG. 1 is an image of a tissue section stained with a compositioncomprising solely the DAB chromogen solution and FIG. 2 is an image of atissue section stained with a composition comprising the DAB chromogensolution and the disclosed polymer (e.g. 2 wt % Dextran Sulfate, Mw=9-20kDa, no PEG).

A. Chromogens

The disclosed composition may comprise one or more chromogens. Presentlydisclosed embodiments are particularly directed to forming compositionscomprising diaminobenzidine (DAB), and/or derivatives thereof. DABchromogens, such as 3,3′-diaminobenzidine, have been used for some timefor various staining procedures, such as immunoblotting andimmunohistochemical staining. The amount of DAB chromogen used fordisclosed embodiments can vary, but functionally is used in amountssufficient to provide an acceptable stained tissue sample, such as anamount of from greater than 0.05 mM to about 100 mM, more typically fromgreater than 0.1 mM to about 15 mM, even more typically from about 1 mMto about 11 mM, with working embodiments typically comprising from about5 to about 5.5 mM DAB chromogen, and/or derivatives thereof.

B. Polymers

Various polymers can be used in the disclosed composition. A suitablepolymer contemplated by the present disclosure typically is awater-soluble polymer comprising a polymeric backbone and at least one,and typically plural functional groups that are effective to reduce orsubstantially preclude precipitation of the DAB chromogen, and/orderivatives thereof. Reactive chemical initiators used in connectionwith polymer synthesis will often leave functional groups on theterminal positions of polymeric chains. While these groups can affectthe solubility and functionality of the polymer, especially for lowmolecular weight polymers, the functionality described herein relates tothe functional groups spanning the polymer backbones. In illustrativeembodiments, polymers having been initiated with reactive initiatorswith functional groups providing additional operability within the scopeof the current disclosure are included in the disclosed composition.

1. Polymeric Backbones

Certain embodiments of disclosed polymers include a polymeric backbone.The polymeric backbone may be water soluble or substantially waterinsoluble. In embodiments wherein the polymeric backbone itself issubstantially water insoluble, the entire polymer (e.g. polymericbackbone in combination with suitable functional groups) should besubstantially water soluble. Particular disclosed embodiments concernpolymeric backbones having either one of Formulas 1, 2, and 3,illustrated below.

With reference to Formula 1, R¹ may be selected from aliphatic, aryl,heteroaryl, and a heteroatom-containing moiety. In one embodiment, R¹may further comprise any of the suitable polymeric functional groupsdisclosed herein. The heteroatom containing moiety may be selected fromester, acid, and amide. R² may be selected from hydrogen, aliphatic,heteroaliphatic, aryl, or heteroaryl. With reference to Formulas 3 and2, each Y independently may be selected from oxygen, sulfur, and NR^(a)wherein R^(a) is selected from hydrogen, aliphatic, aryl, heteroaryl,and heteroaliphatic. R³ may be selected from any of the suitablepolymeric functional groups disclosed herein, and n ranges from 1 toabout 100.

In one embodiment, the polymer may have a Formula 4 or 5, illustratedbelow.

With reference to Formulas 4 and 5, R² may be selected from hydrogen,aliphatic, heteroaliphatic, aryl, or heteroaryl and R³ may be a polymerfunctional group selected from a sulfonate, an amine, a carboxyl group,a carboxylate, or combinations thereof. R⁴ may be selected fromhydrogen, aliphatic, and (CH₂)_(q)NR^(a)R^(b)R^(c) wherein R^(a), R^(b),and R^(c) independently are selected from hydrogen, aliphatic, aryl, andcombinations thereof, and q ranges from 1 to about 10; and n ranges from1 to about 100.

Furthermore, any one of the disclosed polymeric backbones may becombined with another polymeric backbone in order to produce a hybridpolymeric backbone.

Particular disclosed embodiments concern polymeric backbones selectedfrom dextrans, polystyrene, polyvinyl derivatives, polymethacrylatederivatives, polyacrylamide derivatives, polysaccharide derivatives,polypeptide derivatives, nucleic acid and nucleic acid backbonederivatives, polystyrene/maleic acid co-polymers,poly(acrylamide/methacrylate) co-polymers, and the like.

Examples of suitable water-soluble moieties include dextran polymers.Dextrans are readily soluble in water, salt, and electrolyte containingcompositions, and pH has a negligible effect on solubility. Concentrateddextran solutions, such as solutions comprising greater than 50% (w/v)can be prepared. A person of ordinary skill in the art will recognizethat dextrans have various molecular weights.

Other polymeric backbones can be based on less soluble, or substantiallynon-soluble backbones, as long as the polymeric material used to formdisclosed compositions is itself substantially water soluble. Examplesof this class of backbones include aryl backbones, such as exemplifiedby styrene-type polymeric materials.

2. Suitable Polymeric Functional Groups

Suitable functional groups include any functional groups that facilitatemaintaining a DAB chromogen, and/or derivatives thereof, in solutionduring and after product formulation. By way of example, suitablefunctional groups include carboxylate, sulfonate (R—SO₂O⁻), sulfate (SO₄²⁻), amine, including primary, secondary, tertiary and quaternaryamines, substituted amines, and combinations of such functional groups.An amine functional group may be selected from aliphatic amines,particularly amines functionalized with one or more hydrogen atoms orone or more lower alkyl groups, such as methyl, ethyl, propyl, and butylgroups; aryl amines, such as amines functionalized with one or more arylgroups, particularly phenyl; heteroaryl amines; and combinationsthereof. In one embodiment, the polymer may be a polyalkyleneamine

3. Exemplary Polymers

The following polymers are meant only to be exemplary of classes andspecies of polymers suitable for forming the disclosed composition andare not intended to be limiting. Sulfonate and sulfate polymerssubstantially reduce or prevent DAB sulfate precipitation apparently byforming a water-soluble polymer complex with DAB, and/or derivativesthereof. The effect of the sulfate and sulfonate polymers on DABchromogen tissue staining also may be controlled by varying the polymersize, such as weight average molecular weight (Mw) and/or number averagemolecular weight (Mn) and/or the polymer wt % concentration. DABchromogen tissue staining was reduced and hematoxylin backgroundstaining increased with larger Mw polymers. For example, FIGS. 3-8collectively illustrate the effects of exemplary working embodiments onDAB deposition with increasing polystyrene sulfonate (PSS) polymer Mw.FIG. 3 is a control image of a stained tissue section produced using acomposition comprising solely a DAB chromogen solution (e.g. anOPTIVIEW® DAB solution, Ventana Medical Systems, Inc. Tucson Ariz.,hereinafter referred to as “Ventana”). FIGS. 4-8 are images of stainedtissue sections produced using a composition comprising a DAB chromogensolution and PSS polymer having molecular weights of about 1.5 kDa (FIG.4), 5.1 kDa (FIG. 5), 7.5 kDa (FIG. 6), 16 kDa (FIG. 7), and 35 kDa(FIG. 8). All polymers used in these exemplary embodiments were used ata polymer concentration of 5 wt %. As illustrated in FIGS. 4-8, highermolecular weight derivatives of PSS polymers lead to decreased DABstaining and increased hematoxylin background staining. In exemplaryembodiments, low molecular weight dextran sulfate and polystyrenesulfate address DAB sulfate precipitation with a minimal impact on DABtissue staining.

Amine polymers also are useful for reducing, or substantiallypreventing, DAB sulfate precipitation. In one embodiment, amine polymersmay be used to completely inhibit precipitation of DAB, and/orderivatives thereof. Primary, secondary, tertiary and quaternary aminepolymers are contemplated by the present disclosure. Amine polymers donot have the same impact on DAB tissue staining as sulfate and sulfonatepolymers. Without being bound to a particular theory of operation, it iscurrently believed that polymer concentration can increase viscosity andtherefore can affect DAB tissue staining Amine polymers maysubstantially reduce or prevent DAB sulfate precipitation by forming awater-soluble polymer complex between the cationic polymer and sulfateanions, thereby reducing the concentration of free sulfate available forcomplexing with DAB and forming the insoluble DAB sulfate complex.Tissue staining can also be affected by amine polymer solubility, whichcan be an issue at neutral pH in reaction buffer for particularembodiments of amine polymers, such as Mw=15 kDa polyallylamine.Suitable exemplary polymers of amines include, but are not limited to,polyethyleneimine (linear or branched), dextran DEAE andpoly(2-methylacryloxethyltrimethylammonium bromide) polymers.

In one embodiment, the composition comprises a chromogen, or chromogens,and an aminodextran polymer. FIGS. 9-12 collectively illustrate theresults of certain working embodiments using the disclosed composition.FIG. 9 is an image illustrating results obtained with a control whereinonly the DAB chromogen solution was applied to a tissue sample. FIGS.10-12 are images of stained tissue sections wherein a compositioncomprising the DAB chromogen solution and 4 wt % of aminodextran havingvarying molecular weights was added to the tissue section. FIG. 10illustrates results obtained with a Mw=10 kDa aminodextran polymer, FIG.11 concerns a Mw=40 kDa aminodextran polymer, and FIG. 12 illustratesresults obtained using a Mw=70 kDa aminodextran polymer. Polymerconcentration (wt %) of the aminodextran-based polymers typically hasless impact on tissue staining; however, optimization of the polymerconcentration may be carried out in order to promote polymer solubilityand DAB sulfate precipitate prevention.

In other disclosed embodiments, DAB chromogen solutions containingvarying polymer concentrations, such as 0.05 to 3 wt %, of linearpolyethyleneimine (PEI, 2.5 kDa free base or corresponding 4 kDa HClsalt) can be used to address DAB sulfate precipitation. This disclosedembodiment may be used with DAB chromogen solutions comprising up toabout 4 mM of an antioxidant stabilizer, such as sodium metabisulfiteand typically will not negatively impact DAB tissue staining. Otherclasses of amine polymers, such as dextran DEAE and aminodextran, werefound to significantly reduce DAB sulfate precipitation with up to about1 mM of an antioxidant stabilizer, such as sodium metabisulfite, withoutnegatively impacting DAB tissue staining; however, these particulardisclosed embodiments typically provide minimum protection against DABsulfate precipitation at about 4 mM sodium metabisulfite. Otherdisclosed embodiments of amine polymers include branched PEI andpolyallylamine, which may be used to prevent precipitation at stabilizerlevels of about 1 mM to about 4 mM. However, in one embodiment, theseamine polymers may not produce desired DAB staining of the tissuesample.

Based on the above, an exemplary list of particular species of polymericmaterials suitable for the present invention includes dextran sulfate,polystyrene sulfonate, polystyrene sulfonate maleic acid co-polymer,linear polyethyleneimine, dextran DEAE, aminodextran,poly(2-vinyl-1-methylpyridinium bromide),poly(2-methylacryloxethyltrimethylammonium bromide),poly(acrylamide/2-methylacryloxethyltrimethylammonium bromide), andcombinations thereof.

C. DAB/Polymer Ratio, Molecular Weights, and Concentrations

In one embodiment, low molecular weight polymers tend to have less of animpact on DAB staining. The ability to influence counterstaining (e.g.hematoxylin counterstaining) may also be controlled by manipulatingpolymer wt % and molecular weight. Particular disclosed embodimentsconcern a composition comprising a DAB chromogen solution comprising DABchromogen, and/or derivatives thereof, in a concentration ranging fromgreater than 0.1 mM to about 100 mM, more typically from greater than0.5 mM to about 15 mM, even more typically from about 1 mM to about 11mM, with working embodiments typically comprising from about 5 to about11 mM of DAB, and/or derivatives thereof. Disclosed embodiments of thecomposition also comprise one or more of the disclosed polymers orequivalents thereof wherein the polymer has a molecular weight rangingfrom greater than 1 kDa to about 500 kDa, more typically from about 1kDa to about 250 kDa, even more typically from about 1 kDa to about 100kDa. The polymer has a polymer concentration ranging from greater than0.01 to about 10 weight percent, more typically from about 0.1 to about7 weight percent.

In disclosed embodiments where the polymer is a dextran sulfate polymer,the molecular weight of the polymer will typically range from about 5kDa to about 30 kDa at a polymer concentration of from about 2 to about5 weight percent. Even more typically, the dextran sulfate polymer has amolecular weight ranging from about 6.5 kDa to about 20 kDa at a polymerconcentration of from about 2 to about 5 weight percent. If the polymeris a polystyrene sulfonate polymer, then typical embodiments will have amolecular weight ranging from about 1 kDa to about 40 kDa at a polymerconcentration of from about 2 to about 5 weight percent. Even moretypically, the polystyrene sulfonate polymer will have a molecularweight ranging from about 1 kDa to about 35 kDa at a polymerconcentration of from about 2 to about 5 weight percent. Exemplaryworking embodiments used a polystyrene sulfonate polymer having amolecular weight of about 1.5 kDa and a polymer concentration of 2weight percent. If the composition comprises a polystyrene sulfonatemaleic acid co-polymer, then the polymer typically will have a molecularweight of greater than 0 kDa to about 20 kDa and a polymer concentrationof greater than 2 to about 5 weight percent. If the compositioncomprises a polyvinvylsulfonate polymer, then the polymer typically willhave a molecular weight of from about 4 to about 6 kDa and a polymerconcentration of greater than 2 to about 5 weight percent.

If the composition comprises an aminodextran polymer, then the polymertypically will have a molecular weight ranging from about 10 kDa toabout 70 kDa at a polymer concentration of from greater than 0.1 toabout 10 weight percent, more typically from about 0.25 to about 5weight percent. If the composition comprises a dextran DEAE polymer,then the polymer typically will have a molecular weight ranging fromgreater than 1 kDa to about 500 kDa and a polymer concentration rangingfrom greater than 0.1 to about 7 weight percent. In other disclosedembodiments, the composition may comprise a polyallylamine polymerhaving a molecular weight of greater than 1 kDa to about 15 kDa and apolymer concentration ranging from greater than 0.01 to about 5 weightpercent. In exemplary embodiments, the composition comprises apolyethyleneimine polymer having a molecular weight of about 2.5 kDa anda polymer concentration of about 0.15 weight percent. Thepolyethyleneimine polymer may be selected from a free base polymer or asalt, wherein the salt typically is a 4 kDa HCl salt of the samecorresponding 2.5 kDa polyethyleneimine free base polymer.

D. DAB Base Formulations

One embodiment of a DAB base solution comprises DAB chromogen, one ormore enhancers, and a stabilizer. In one embodiment, the DAB basesolution comprises a DAB chromogen, and/or derivatives thereof, having aconcentration ranging from greater than 0.1 mM to about 15 mM, moretypically from greater than 0.1 mM to about 11 mM, an imidazole enhancerhaving a concentration ranging from greater than 0.01 mM to about 15 mM,a 2-hydroxypyridine enhancer having a concentration ranging from greaterthan 0.01 mM to about 15 mM, and a sodium metabisulfite stabilizerhaving a concentration ranging from greater than 0.01 mM to about 2 mM.Exemplary base solutions comprise 5.5 mM DAB, 10 mM imidazole, 10 mM2-hydroxypyridine, and 1 mM sodium metabisulfite, with a pH of 2.5±0.1.In one embodiment, these compositions were tested using KHSO₄concentrations ranging from 0 to about 10 mM; more typically 0, 1.37,2.75 and 8.0 mM, with DAB sulfate precipitation being induced at 4° C.overnight. In another embodiment, the enhancer is omitted from the DABbase formulation and included in the oxidant formulation, as disclosedherein, in concentrations equivalent to those specified herein.

Certain DAB chromogen compositions are known, and are commerciallyavailable, which do not include a polymeric stabilizer as is used withembodiments disclosed in the present application. For example, VentanaMedical Systems, Inc., provides a DAB chromogen formulation comprising5.5 mM DAB.4HCl, 10 mM imidazole, 10 mM 2-hydroxypyridine, 1 mM sodiummetabisulfite, 5 wt % PEG8000, 0.05% w/v Brij 35, and 750 μM sodiumstannate trihydrate, with a pH of 2.3±0.1.

A person of ordinary skill in the art will recognize that sodiummetabisulfite disproportionates in water to form 2 moles of sodiumbisulfite per mole of sodium metabisultite. Each mole of sodiumbisulfite can form one mole of sodium hydrogen sulfate which can form aninsoluble salt complex with DAB. In one embodiment, DAB sulfate wasdetermined to most likely be a DAB monosulfate salt by analysis.

Certain disclosed embodiments of a new DAB chromogen compositiondeveloped by Ventana Medical Systems, Inc. comprise 5.5 mM DAB.4HCl, 10mM imidazole, 10 mM 2-hydroxypyridine, 1 mM sodium metabisulfite, 0.15wt % 2.5 kDa linear PEI (free base or 4 kDa HCl salt polymers), and0.05% w/v Brij 35, with a pH of 2.3±0.1. Other embodiments of DABchromogen compositions comprise 0.15 wt % 2.5 kDa linear PEI (free baseor 4 kDa HCl salt), 5-7 wt % 500 kDa dextran DEAE, 2 wt % 6.5-10 kDadextran sulfate and 2 wt %˜1.5 kDa PSS polymers.

IV. Method of Using the Composition

The present disclosure also concerns embodiments of a method for usingDAB chromogen compositions disclosed herein. In one embodiment, themethod may comprise providing a tissue sample comprising an epitope;exposing the tissue sample to a labeled specific binding moiety and alabeled enzyme; exposing the tissue sample to a composition comprisingDAB chromogen, or derivative thereof, in an amount effective for atissue staining procedure, an effective amount of stabilizer, andpolymer in an amount effective to reduce or substantially prevent DABprecipitation relative to a composition that does not include thepolymer; exposing the tissue sample to an oxidant and a counterstain;and detecting the epitope.

In one embodiment, the tissue sample may be any tissue sample capable ofbeing analyzed using colorimetric detection. Particular disclosedembodiments concern using a formalin-fixed, paraffin-embedded tissuesample. The labeled specific binding moiety may be labeled with a firstmember of a specific binding pair, such as a hapten (e.g. an oxazole, apyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, abenzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin,a cyclolignan, a heterobiaryl, an azo aryl, or a benzodiazepine). In oneembodiment, the hapten is selected from HQ, DIG, DNP, TS, NP, DCC, andbiotin. Exemplary haptens and conjugates comprising haptens aredisclosed in U.S. Pat. No. 7,695,929, which is incorporated herein byreference. The specific binding moiety may be selected from an antibodyor a nucleic acid (e.g. DNA or RNA). In one embodiment, the labeledenzyme is an enzyme that is conjugated with a second member of aspecific binding pair, such as an antibody or avidin (e.g.streptavidin). The enzyme may be a peroxidase, such as horseradishperoxidase.

The tissue sample may be exposed to the disclosed composition using avariety of different techniques. In one embodiment, the tissue sample isexposed to the composition by adding separate solutions of the DABchromogen and the polymer to the tissue sample simultaneously, bysequentially adding separate solutions of the DAB chromogen and thepolymer to the tissue sample, or by adding a solution comprising DABchromogen and the polymer to the tissue sample. The composition maycomprise any of the disclosed components described herein. Afteraddition of the disclosed composition, an oxidant, such as hydrogenperoxide, may be added to the tissue sample. Also, a counterstain, suchas hematoxylin may be added to the tissue sample. An epitope within thetissue sample may then be detected using colorimetric detection. Thedisclosed method may be performed manually or may be partially or fullyautomated.

V. Kits

The present disclosure also contemplates embodiments of a kit comprisingthe disclosed composition. In one embodiment, the disclosed kit maycomprise DAB chromogen, or derivative thereof; an enhancer selected fromimidazole, 2-hydroxypyridine, and combinations thereof; an antioxidantstabilizer, such as sodium metabisulfite; and polymer capable ofreducing or preventing DAB sulfate precipitation relative to acomposition that does not include the polymer. The reagents may bepackaged separately, or two or more reagents may be in the samesolution.

VI. Samples and Targets

The disclosed composition may be used to detect one or more targets in asample. Samples include biological components and generally aresuspected of including one or more target molecules of interest. Targetmolecules can be on the surface of cells and the cells can be in asuspension, or in a tissue section. Target molecules can also beintracellular and detected upon cell lysis or penetration of the cell bya probe. One of ordinary skill in the art will appreciate that themethod of detecting target molecules in a sample will vary dependingupon the type of sample and probe being used. Methods of collecting andpreparing samples are known in the art.

Samples for use in the embodiments of the method and with thecomposition disclosed herein, such as a tissue or other biologicalsample, can be prepared using any method known in the art by of one ofordinary skill. The samples can be obtained from a subject for routinescreening or from a subject that is suspected of having a disorder, suchas a genetic abnormality, infection, or a neoplasia. The describedembodiments of the disclosed method can also be applied to samples thatdo not have genetic abnormalities, diseases, disorders, etc., referredto as “normal” samples. Such normal samples are useful, among otherthings, as controls for comparison to other samples. The samples can beanalyzed for many different purposes. For example, the samples can beused in a scientific study or for the diagnosis of a suspected malady,or as prognostic indicators for treatment success, survival, etc.

Samples can include multiple targets that can be specifically bound by aprobe or reporter molecule. The targets can be nucleic acid sequences orproteins. Throughout this disclosure when reference is made to a targetprotein it is understood that the nucleic acid sequences associated withthat protein can also be used as a target. In some examples, the targetis a protein or nucleic acid molecule from a pathogen, such as a virus,bacteria, or intracellular parasite, such as from a viral genome. Forexample, a target protein may be produced from a target nucleic acidsequence associated with (e.g., correlated with, causally implicated in,etc.) a disease.

A target nucleic acid sequence can vary substantially in size. Withoutlimitation, the nucleic acid sequence can have a variable number ofnucleic acid residues. For example a target nucleic acid sequence canhave about 10 nucleic acid residues, or about 20, 30, 50, 100, 150, 500,1000 residues. Similarly, a target polypeptide can vary substantially insize. Without limitation, the target polypeptide will include epitopethat binds to a peptide specific antibody, or fragment thereof. In someembodiments that polypeptide can include two epitopes that bind to apeptide specific antibody, or fragment thereof.

In specific, non-limiting examples, a target protein is produced by atarget nucleic acid sequence (e.g., genomic target nucleic acidsequence) associated with a neoplasm (for example, a cancer). Numerouschromosome abnormalities (including translocations and otherrearrangements, amplification or deletion) have been identified inneoplastic cells, especially in cancer cells, such as B cell and T cellleukemias, lymphomas, breast cancer, colon cancer, neurological cancersand the like. Therefore, in some examples, a portion of the targetmolecule is produced by a nucleic acid sequence (e.g., genomic targetnucleic acid sequence) amplified or deleted in a subset of cells in asample.

Oncogenes are known to be responsible for several human malignancies.For example, chromosomal rearrangements involving the SYT gene locatedin the breakpoint region of chromosome 18q11.2 are common among synovialsarcoma soft tissue tumors. The t(18q11.2) translocation can beidentified, for example, using probes with different labels: the firstprobe includes FPC nucleic acid molecules generated from a targetnucleic acid sequence that extends distally from the SYT gene, and thesecond probe includes FPC nucleic acid generated from a target nucleicacid sequence that extends 3′ or proximal to the SYT gene. When probescorresponding to these target nucleic acid sequences (e.g., genomictarget nucleic acid sequences) are used in an in situ hybridizationprocedure, normal cells, which lack a t(18q11.2) in the SYT gene region,exhibit two fusion (generated by the two labels in close proximity)signals, reflecting the two intact copies of SYT. Abnormal cells with at(18q11.2) exhibit a single fusion signal.

In other examples, a target protein produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) is selected thatis a tumor suppressor gene that is deleted (lost) in malignant cells.For example, the p16 region (including D9S1749, D9S1747, p16(INK4A),p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21is deleted in certain bladder cancers. Chromosomal deletions involvingthe distal region of the short arm of chromosome 1 (that encompasses,for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), andthe pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (thatencompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, andGLTSCR1) are characteristic molecular features of certain types of solidtumors of the central nervous system.

The aforementioned examples are provided solely for purpose ofillustration and are not intended to be limiting. Numerous othercytogenetic abnormalities that correlate with neoplastic transformationand/or growth are known to those of ordinary skill in the art. Targetproteins that are produced by nucleic acid sequences (e.g., genomictarget nucleic acid sequences), which have been correlated withneoplastic transformation and which are useful in the disclosed methods,also include the EGFR gene (7p12; e.g., GENBANK™ Accession No.NC_000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21;e.g., GENBANK™ Accession No. NC_000008, nucleotides128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene(8p22; e.g., GENBANK™ Accession No. NC_000008, nucleotides19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC_000013,nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK™ AccessionNo. NC_000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24;e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No.NC_000012, complement, nucleotides 56196638-56200567), FUS (16p11.2;e.g., GENBANK™ Accession No. NC_000016, nucleotides 31098954-31110601),FKHR (13p14; e.g., GENBANK™ Accession No. NC_000013, complement,nucleotides 40027817-40138734), as well as, for example: ALK (2p23;e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™Accession No. NC_000011, nucleotides 69165054..69178423), BCL2 (18q21.3;e.g., GENBANK™ Accession No. NC_000018, complement, nucleotides58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC_000003,complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31;e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No.NC_000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3;e.g., GENBANK™ Accession No. NC_000021, complement, nucleotides41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No.NC_000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3;e.g., GENBANK™ Accession No. NC_000007, complement, nucleotides13897379-13995289), EWS (22q12.2; e.g., GENBANK™ Accession No.NC_000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g.,GENBANK™ Accession No. NC_000011, nucleotides 128069199-128187521), PAX3(2q35-q37; e.g., GENBANK™ Accession No. NC_000002, complement,nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™Accession No. NC_000001, nucleotides 18830087-18935219), PTEN (10q23.3;e.g., GENBANK™ Accession No. NC_000010, nucleotides 89613175-89716382),AKT2 (19q13.1-q13.2; e.g., GENBANK™ Accession No. NC_000019, complement,nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ AccessionNo. NC_000001, complement, nucleotides 40133685-40140274), REL(2p13-p12; e.g., GENBANK™ Accession No. NC_000002, nucleotides60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No.NC_000005, complement, nucleotides 149413051-149473128).

In other examples, a target protein is selected from a virus or othermicroorganism associated with a disease or condition. Detection of thevirus- or microorganism-derived target nucleic acid sequence (e.g.,genomic target nucleic acid sequence) in a cell or tissue sample isindicative of the presence of the organism. For example, the targetpeptide, polypeptide or protein can be selected from the genome of anoncogenic or pathogenic virus, a bacterium or an intracellular parasite(such as Plasmodium falciparum and other Plasmodium species, Leishmania(sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardialamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesiaspecies).

In some examples, the target protein is produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) from a viralgenome. Exemplary viruses and corresponding genomic sequences (GENBANK™RefSeq Accession No. in parentheses) include human adenovirus A(NC_001460), human adenovirus B (NC_004001), human adenovirus C(NC_001405), human adenovirus D (NC_002067), human adenovirus E(NC_003266), human adenovirus F (NC_001454), human astrovirus(NC_001943), human BK polyomavirus (V01109; GI:60851) human bocavirus(NC_007455), human coronavirus 229E (NC_002645), human coronavirus HKU1(NC_006577), human coronavirus NL63 (NC_005831), human coronavirus OC43(NC_005147), human enterovirus A (NC_001612), human enterovirus B(NC_001472), human enterovirus C (NC_001428), human enterovirus D(NC_001430), human erythrovirus V9 (NC_004295), human foamy virus(NC_001736), human herpesvirus 1 (Herpes simplex virus type 1)(NC_001806), human herpesvirus 2 (Herpes simplex virus type 2)(NC_001798), human herpesvirus 3 (Varicella zoster virus) (NC_001348),human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC_007605),human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334),human herpesvirus 5 strain AD169 (NC_001347), human herpesvirus 5 strainMerlin Strain (NC_006273), human herpesvirus 6A (NC_001664), humanherpesvirus 6B (NC_000898), human herpesvirus 7 (NC_001716), humanherpesvirus 8 type M (NC_003409), human herpesvirus 8 type P(NC_009333), human immunodeficiency virus 1 (NC_001802), humanimmunodeficiency virus 2 (NC_001722), human metapneumovirus (NC_004148),human papillomavirus-1 (NC_001356), human papillomavirus-18 (NC_001357),human papillomavirus-2 (NC_001352), human papillomavirus-54 (NC_001676),human papillomavirus-61 (NC_001694), human papillomavirus-cand90(NC_004104), human papillomavirus RTRX7 (NC_004761), humanpapillomavirus type 10 (NC_001576), human papillomavirus type 101(NC_008189), human papillomavirus type 103 (NC_008188), humanpapillomavirus type 107 (NC_009239), human papillomavirus type 16(NC_001526), human papillomavirus type 24 (NC_001683), humanpapillomavirus type 26 (NC_001583), human papillomavirus type 32(NC_001586), human papillomavirus type 34 (NC_001587), humanpapillomavirus type 4 (NC_001457), human papillomavirus type 41(NC_001354), human papillomavirus type 48 (NC_001690), humanpapillomavirus type 49 (NC_001591), human papillomavirus type 5(NC_001531), human papillomavirus type 50 (NC_001691), humanpapillomavirus type 53 (NC_001593), human papillomavirus type 60(NC_001693), human papillomavirus type 63 (NC_001458), humanpapillomavirus type 6b (NC_001355), human papillomavirus type 7(NC_001595), human papillomavirus type 71 (NC_002644), humanpapillomavirus type 9 (NC_001596), human papillomavirus type 92(NC_004500), human papillomavirus type 96 (NC_005134), humanparainfluenza virus 1 (NC_003461), human parainfluenza virus 2(NC_003443), human parainfluenza virus 3 (NC_001796), human parechovirus(NC_001897), human parvovirus 4 (NC_007018), human parvovirus B19(NC_000883), human respiratory syncytial virus (NC_001781), humanrhinovirus A (NC_001617), human rhinovirus B (NC_001490), humanspumaretrovirus (NC_001795), human T-lymphotropic virus 1 (NC_001436),human T-lymphotropic virus 2 (NC_001488).

In certain examples, the target protein is produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) from an oncogenicvirus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV,e.g., HPV16, HPV18). In other examples, the target protein produced froma nucleic acid sequence (e.g., genomic target nucleic acid sequence) isfrom a pathogenic virus, such as a Respiratory Syncytial Virus, aHepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARSvirus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or aHerpes Simplex Virus (HSV).

VII. Working Embodiments

The following examples are provided to exemplify certain details ofworking embodiments. A person of ordinary skill in the art willappreciate that the present invention is not limited to those specificfeatures exemplified by these examples.

A. General Procedures

1. Polymer Screening

Polymers were screened for their impact on DAB sulfate precipitationformation by combining an enhanced DAB base solution containing a setamount of the polymer with select molar ratios of potassium hydrogensulfate, KHSO₄, relative to DAB. The enhanced DAB base solution usedcomprised 5.5 mM DAB.4HCl, 10 mM imidazole, 10 mM 2-hydroxypyridine, 1mM sodium metabisulfite and 0.05% w/v Brij 35 at pH=2.3±0.1. Thepotassium hydrogen sulfate concentrations screened were 2.75 and 8.0 mM.The solutions were mixed vigorously at room temperature for 15 secondsand then stored at 2-8° C. to induce DAB sulfate precipitation.Solutions were monitored for physical changes (i.e. color orprecipitation). The candidate solutions were analyzed by HPLC before andafter sulfate addition to confirm changes in DAB concentration relativeto 2-hydroxypyridine as an internal standard.

2. Immunohistochemistry Staining Procedure

All immunohistochemistry was performed on a Ventana Benchmark XTautomated staining platform using components of either the VENTANAOPTIVIEW® DAB (Ventana Medical Systems, Inc. Catalog #760-700) or iViewDAB (Ventana Medical Systems, Inc. Catalog #760-091) detection kitsfollowing suggested product insert protocols. Antigen detection wasperformed using either CONFIRM® anti-Ki67 (30-9) antibody (Ventana#790-4286) or anti-CD10 (SP67) antibody (Ventana #790-4056) by addingone drop of either antibody and incubating at 37° C. for 16 minutes. DABIHC Detection was achieved by adding one drop of the candidate DABchromogen solution, followed by one drop of the appropriate H₂O₂solution and co-incubating for 8 minutes. Slides were counterstainedwith Hematoxylin II (Ventana #790-2208) for 4 minutes, followed byBluing (Ventana 760-2037) for four minutes. The slides were rinsed witha detergent water mixture, dehydrated through gradient alcohol andxylene baths and coverslipped.

3. HPLC Analytical Method

HPLC analysis was performed using the method below and FIG. 13illustrates representative HPLC spectra obtained using this exemplarymethod.

Column: Waters Xbridge RP-C18 Column 4 6×150 mm (5 p) [Phenomenex C18Security guard column]Eluents: A: deionized water, C: acetonitrile, D: 100 mM aq. ammoniumacetate (pH=5.0)Flow rate: 1 mL/minGradient profile:

Time (min) % A % C % D 0 83 1 16 10 4 80 16 12 4 80 16 13 83 1 16 16 831 16Injection volume: 3 μL of DAB chromogen solution without dilutionDetection wavelength: 260 nmRetention time: DAB (4.94 min), 2-hydroxypyridine (3.65 min)

EXAMPLES Example 1

Candidate polymers were screened for their impact on DAB sulfateprecipitation formation by combining an enhanced DAB base solutioncontaining a set amount of the polymer with select molar ratios ofpotassium hydrogen sulfate, KHSO₄, relative to DAB. The enhanced DABbase solution used comprised 5.5 mM DAB.4HCl, 10 mM imidazole, 10 mM2-hydroxypyridine, 1 mM sodium metabisulfite and 0.05% w/v Brij 35 atpH=2.3±0.1. The potential potassium hydrogen sulfate screeningconcentrations were 0, 1.37, 2.75 and 8.0 mM. The solutions were mixedvigorously at room temperature for 15 seconds and then stored at 2-8° C.to induce DAB sulfate precipitation. Solutions were monitored forphysical changes (i.e. color or precipitation). The candidate solutionswere analyzed by HPLC before and after sulfate addition to confirmchanges in DAB concentration relative to 2-hydroxypyridine as aninternal standard. A summary of results are shown in Table 2 below.

Exemplary polymers (dextran sulfate and polystyrene sulfonate)substantially inhibited DAB precipitation, and other exemplary polymers(dextran DEAE and 10 kDa aminodextran) also significantly reduced DABprecipitation. For example, FIGS. 14-16 collectively illustrate resultsobtained using exemplary sulfonate and sulfate polymers in comparison toa sampler wherein no polymer is used (FIG. 14). FIG. 15 is an image of atissue section stained using the disclosed composition comprising theDAB solution and 4 wt % polystyrene sulfonate (1.5 kDa) and FIG. 16 isan image of a tissue section stained using the disclosed compositioncomprising the DAB solution and 4 wt % dextran sulfate (6.5-10 kDa).FIGS. 17 and 18 are images illustrating results obtained using a control(FIG. 17) and 5 wt % 15 kDa polyallylamine (FIG. 18). Further HPLCresults of exemplary embodiments of the disclosed composition aresummarized in Table 2.

TABLE 2 HPLC analytical data for candidate polymer stabilized DABchromogen solutions treated with potassium hydrogen sulfate 2.75 mMKHSO₄ 8.0 mM KHSO₄ Enhanced DAB base (5.5 mM DAB) DAB/2HP % Δ DAB/2HP %Δ Standard without KHSO₄ addition 4.49 4.49 Standard - No Polymer Added   2.22 (b) −50.6    0.40 (b) −91.1 PEG8000, 5 wt %    2.40 (b) −46.5n/d n/d 9-20 kDa Dextran Sulfate, 5 wt % 4.07 −9.4 3.66 −18.5 6.5-10 kDaDextran Sulfate, 5 wt % 4.33 −3.6 4.20 −6.5 1.53 kDa PolystyreneSulphonate, 5 wt % 4.07 −9.4 3.85 −14.3 5.18 kDa Polystyrene Sulphonate,5 wt % 4.14 −7.8 4.00 −10.9 20 kDa Polystyrene Sulfonate Maleic Acid Co-4.10 −8.7 4.00 −10.9 polymer, 5 wt % 2.5 kDa Linear polyethyleneimine (4kDa HCl salt), 4.50 0.0 4.49 0.0 2 wt % 2.5 kDa Linear polyethyleneimine(free base), 2 wt % 4.51 0.0 4.56 0.0 1.8 kDa Branched Polyethyleneimine(free base), 2   3.82 (a) −14.9   3.82 (a) −14.9 wt % 500 kDa DextranDEAE, 7 wt %     3.89 (a, b) −13.4    1.08 (b) −75.9 10 kDaAminodextran, 5 wt %     3.81 (a, b) −15.1    0.68 (b) −84.9Poly(2-vinyl-1-methylpyridinium bromide), 2 wt %   4.25 (a) −5.3    1.89 (a, b) −57.9 Poly(2-methylacryloxethyltrimethylammonium 4.01−10.7     1.00 (a, b) −77.7 bromide), 2 wt %Poly(acrylamide/2-methylacryloxethyl-    1.87 (b) −58.3    0.30 (b)−93.3 trimethylammonium bromide) (80:20), 2 wt % (a): New HPLC peak(s)present not yet identified, (b): DAB sulfate precipitate observed

HPLC analysis showed DAB solutions with and without PEG8000 both lost asimilar amount of DAB from solution after addition of addition of 2.75mM KHSO₄. Polyols and alcohols have been reported to reduce DAB sulfateprecipitation. A non-volatile alcohol additive, such as 1,2-propanediol,failed to inhibit DAB sulfate precipitation. Dextran polymers reducedbut did not completely inhibit DAB sulfate precipitation. Other polymerclasses such as polyvinylalcohol (PVA), polyacrylamide (PAA),polyvinylpyrrolidinone (PVP), poly(propylene glycol) (PPG) were testedand failed to inhibit DAB sulfate precipitation.

Example 2

This particular example concerns using amine polymers in the disclosedcomposition. Amine polymers had a wide range of influence on DAB sulfateprecipitate formation Amine polymers are believed to form salts with thesulfate counteranion reducing frequency of DAB sulfate formation and/orhelp reduce the DAB sulfate particle size. DAB sulfate precipitation wasnot observed with DAB chromogen solutions containing linearpolyethyleneimine (PEI), branched PEI or polyallylamine (PAA) polymersupon addition of either 2.75 or 8.0 mM KHSO₄. The influences of linearPEI polymer concentration (wt %) was examined in this process. A 2.5 kDalinear PEI was used as a 4 kDa HCl salt and varied from 0.1 to 5.0 wt %in the chromogen solution (see Table 3). The linear PEI inhibited DABsulfate precipitation over the entire polymer concentration range at2.75 mM KHSO₄. DAB sulfate precipitate was only observed with 8.0 mMKHSO₄ at 0.1 wt % PEI. Linear and branched PEI polymers were examinedfrom 0.8 to 25 kDa in size. The polymer molecular weight did not changethe polymer's effect on inhibiting DAB sulfate precipitation.

TABLE 3 HPLC analytical data for amine polymer stabilized DAB chromogensolutions treated with potassium hydrogen sulfate 2.75 mM 8.0 mM KHSO₄KHSO₄ DAB/ DAB/ Enhanced DAB base (5.5 mM DAB) 2HP % Δ 2HP % Δ Standardwithout KHSO₄ addition 5.03 5.03 5 wt % Linear PEI (4 kDa HCl Salt) 5.030.4 5.06 1.0 4 wt % Linear PEI (4 kDa HCl Salt) 5.04 0.6 5.08 1.4 3 wt %Linear PEI (4 kDa HCl Salt) 5.02 0.2 5.10 1.8 2 wt % Linear PEI (4 kDaHCl Salt) 5.01 0.0 5.05 0.8 1 wt % Linear PEI (4 kDa HCl Salt) 5.02 0.25.03 0.4 0.5 wt % Linear PEI (4 kDa HCl 5.03 0.4 5.05  0.8 * Salt) 0.1wt % Linear PEI (4 kDa HCl 5.04  0.6 * 2.38  −52.5 ** Salt) * A veryminor haze was observed on bottom of vial, but no appreciable DABsulfate ppt. formed. ** DAB sulfate precipitate was observed withextended storage at 4° C.

Aminodextran, dextran DEAE and quaternary amine polymers helped reduce,but did not completely inhibit, DAB sulfate precipitation. Amine polymerwt % concentration was important for these polymers. Higherconcentrations of dextran DEAE were necessary to control the amount ofDAB sulfate precipitation (see Table 4). The water solubility limit fordextran DEAE under formulation conditions did not allow forconcentrations above 7 wt % of polymer to be examined.

TABLE 4 HPLC analytical data for 500 kDa dextran DEAE polymer stabilizedDAB chromogen solutions treated with potassium hydrogen sulfate 2.75 mMKHSO₄ 500 kDa Dextran DEAE (5.5 mM DAB) DAB/2HP % Δ Base no polymer orKHSO₄ 4.49 n/a Base no polymer 2.40 −47 5 wt % PEG8000 2.40 −47 3 wt %Dextran DEAE 2.75 −39 4 wt % Dextran DEAE 2.94 −35 5 wt % Dextran DEAE3.48 −22 6 wt % Dextran DEAE 3.43 −24 7 wt % Dextran DEAE 3.96 −12

The influence of polymer concentration on DAB IHC staining was examinedusing an iView detection method (tonsil tissue, Ki67). A 2.5 kDa linearPEI was used as a 4 kDa HCl salt and varied from 0.1 to 5.0 wt % in theDAB chromogen solution. Representative images are shown at 20× (seeFIGS. 19-22). More viscous DAB chromogen solutions reduced DAB IHCstaining intensity and tissue coverage. Higher viscosity chromogensolutions are well known to reduce enzyme reaction rates in plate-basedassays Amine polymer solubility can be an issue at neutral pH in VentanaReaction Buffer (i.e. 15 kDa polyallylamine,) which greatly affected theintensity and coverage of both DAB IHC and hematoxylin staining.

The influence of aminodextran polymer molecular weight on DAB IHCstaining was examined using an iView detection method (tonsil tissue,Ki67). Aminodextran polymer-stabilized DAB chromogen solutions wereprepared using 4 wt % of different molecular weight polymers.Representative images are shown at 10× (see FIGS. 9-12). DAB IHCstaining intensity was not significantly affected by increasing polymermolecular weight. The aminodextran polymer was soluble in water and hadno effect on solution viscosity at 4 wt %.

Amine polymer stabilized DAB chromogen solutions can be used withoutimpacting DAB IHC tissue staining. Polymer wt % can be controlled tohelp inhibit DAB sulfate precipitation and control solution viscosity toreduce deleterious tissue staining effects. Exemplary workingembodiments in this class include a 2.5 kDa linear PEI (2.5 kDa freebase or 4 kDa HCl salt) and to a lesser extent 500 kDa dextran DEAE.Representative images are shown in FIGS. 23-25. These figures illustrateresults of a tissue section stained with stabilized DAB chromogensolutions comprising no polymer (FIG. 23); 7 wt % 500 kDa dextran DEAE(FIG. 24); and 2 wt % 2.5 kDa linear PEI (FIG. 25). Dextran DEAE canhave a minor influence on hematoxylin staining intensity, but it is muchmore subtle than observed with dextran sulfate.

Polymer stabilized DAB (OPTIVIEW® DAB solution) chromogen solutions werestained on tonsil tissue for Ki67 and CD10 antigen markers. Twodifferent tissue blocks were examined for each antigen marker. Fiveserial section samples were stained for each condition. Each sample wasevaluated by a qualified pathologist for DAB IHC staining intensity, IHCbackground, and hematoxylin counterstain quality. Each sample wascompared to the commercially available DAB reagent without polymer(OPTIVIEW® DAB solution). A summary of the IHC staining results can befound in Table 5. No differences were observed for IHC backgroundstaining between chromogen samples. Subtle differences were observed inthe hematoxylin counterstain between chromogen samples, but thecounterstain hue and intensities were judged to be acceptable. The 2.5kDa linear polyethylenimine free base and corresponding 4 kDa linear PEIHCl salt stained equivalently or better than the commercially availablechromogen reagent on all samples except on Ki67 sample set. The 4 kDalinear PEI HCl salt stained slightly better than the 2.5 kDa linear PEIfree base for CD10.

TABLE 5 IHC Detection of Ki67 and CD10 Antigens on Tonsil using PolymerStabilized DAB Chromogen Solutions OptiView DAB IHC OptiView DAB IHCStaining Intensity Staining Intensity (Ki67, Tonsil) (CD10, Tonsil)Tissue Block Tissue Block Tissue Block Tissue Block 352-10-23 397-10-12357-10-10 386-10-7 ave. std. ave. std. ave. std. ave. std. DAB + PolymerStabilizer score dev. score dev. score dev. score dev. A. Commerciallyavailable DAB 2.4 0.5 3.0 0.0 1.8 0.4 1.6 0.5 B. A with no PEG orstannate 2.4 0.5 3.0 0.0 1.4 0.9 1.8 0.4 B. with 0.15 wt % linear PEI2.2 0.4 3.0 0.0 2.0 0.0 2.0 0.0 (4 kDa HCl salt) B. with 0.15 wt %Linear PEI 2.2 0.4 3.0 0.0 1.8 0.4 1.8 0.4 (2.5 kDa free base) B. with2.0 wt % Dextran Sulfate 1.8 0.4 2.0 0.0 1.4 0.5 1.4 0.5 (6.5-10 kDa) B.with 2.0 wt % Polystyrenesulfonate 2.8 0.4 2.6 0.5 1.2 0.4 1.0 0.0 (1kDa) B. with 7.0 wt % Dextran DEAE (500 1.1 0.5 2.0 0.7 1.4 0.5 1.0 0.5kDa)

The amine polymer stabilized DAB chromogen solutions were thermallystressed at 45° C. to examine chromogen stability in the presence of thenew polymers. DAB chromogen solutions stabilized with 4 kDa linear PEIHCl salt polymer (2.5 kDa free base equiv.) demonstrated the greatestchromogen stability (see Table 6 and 7) for those polymers tested. DABChromogen solutions with <1 wt % 4 kDa linear PEI HCl salt polymerdemonstrated stability greater than the base chromogen solution. DABChromogen solutions with 2.5 kDa linear PEI free base polymerdemonstrated similar stability to the base chromogen solution. BranchedPEI, dextran DEAE and 4° C. amine polymers formed byproducts with DABdecreasing chromogen stability. These byproducts were not isolated orconfirmed by structural analysis.

TABLE 6 HPLC analytical data for polymer stabilized DAB chromogensolutions thermally stressed at 45° C. 1 d - 45° C. 4 d - 45° C. 10 d -45° C. 24 d - 45° C. DAB DAB DAB DAB DAB base + Polymer 2HP % Δ 2HP % Δ2HP % Δ 2HP % Δ No Polymer Standard 4.89 −2.4 4.82 −3.8 4.73 −5.6 3.81−24 5 wt % Linear PEI (4 kDa 4.91 −2.0 4.95 −1.2 4.64 −7.4 3.90 −22 HClSalt) 4 wt % Linear PEI (4 kDa 4.90 −2.2 4.95 −1.2 4.68 −6.6 3.97 −21HCl Salt) 3 wt % Linear PEI (4 kDa 4.86 −3.0 4.97 −0.8 4.72 −5.8 3.70−26 HCl Salt) 1 wt % Linear PEI (4 kDa 5.00 −0.2 4.97 −0.8 4.69 −6.44.31 −14 HCl Salt) 0.5 wt % Linear PEI (4 kDa 5.01   0.0 5.02   0.2 4.77−4.8 4.35 −13 HCl Salt) 0.1 wt % Linear PEI (4 kDa 5.00 −0.2 5.02   0.24.77 −4.8 4.77   −4.8 HCl Salt) 5 wt % Branched PEI (25 2.29 −54**  2.32−54**  0.82 −84**  n/a n/a kDa HCl Salt) 4 wt % Branched PEI (25 2.66−47**  2.65 −47**  1.53 −69**  n/a n/a kDa HCl Salt) 3 wt % Branched PEI(25 2.99 −40**  3.01 −40**  2.05 −59**  n/a n/a kDa HCl Salt) 2 wt %Branched PEI (25 3.26 −35**  3.30 −34**  2.70 −46**  n/a n/a kDa HClSalt) 7 wt % Dextran DEAE (500 4.61  −8.0** 4.51 −10**  4.01 −20**  2.45 −51** kDa) **New HPLC byproduct peak(s) were present in chromogensolution.

TABLE 7 HPLC analytical data for polymer stabilized DAB chromogensolutions thermally stressed at 45° C. 1 d - 45° C. 3 d - 45° C. 9 d -45° C. 23 d - 45° C. DAB DAB DAB DAB DAB base + 2 wt % Polymer 2HP % Δ2HP % Δ 2HP % Δ 2HP % Δ No Polymer Control 4.91  −2.0 4.82  −3.8 4.66 −7.0 3.66 −26.9  Poly(2-vinyl-1-methylpyridinium 4.08 −19** 3.70 −26**2.42 −52** n.d. −100**  bromide) Poly(acrylamide/2- 4.71   −6.0** 4.50−10** 4.33 −14** 3.92 −22** methylacryloxethyl- trimethylammoniumbromide) (80:20) Poly(2-methylacryloxethyltrimethyl- 4.53   −9.6** 4.34−13** 4.14 −17** 2.15 −57** ammonium bromide) Branched PEI Low MW (2.0kDa HCl 3.21 −36** 3.20 −36** 2.98 −41** 1.26 −75** Salt) Branched PEI(1.3 kDa HCl Salt) 3.37 −33** 3.37 −33** 3.29 −34** 2.27 −55** BranchedPEI (1.8 kDa HCl Salt) 3.42 −32** 3.01 −40** 1.21 −76** n.d. −100** Linear PEI (2.5 kDa free amine 4.97  −0.8 4.89  −2.4 4.71  −6.0 3.60−28.1  polymer) **New HPLC byproduct peak(s) were present in chromogensolution.

DAB sulfate precipitation was not observed with DAB chromogen solutionscontaining linear polyethyleneimine (PEI), branched PEI orpolyallylamine (PAA) polymers upon addition of either 2.75 or 8.0 mMKHSO₄. Branched PEI polymers appear to react with DAB forming byproductsobserved in HPLC analysis Aminodextran, dextran DEAE and quaternaryamine polymers reduced but did not completely inhibit DAB sulfateprecipitation. New DAB-polymers byproducts were observed during HPLCanalysis with aminodextran and 4° amine polymers. The proposedDAB-polymer byproducts were not isolated or confirmed by structuralanalysis. Small monomeric like amine materials, namely amino acids orethanolamines (mono-, di- and tri-), did not inhibit DAB sulfateprecipitation.

Example 3

This example concerns particular embodiments of the disclosedcomposition and sulfate and/or sulfonate polymers. Sulfate and sulfonatepolymers are believed to form a DAB-polymer salt complex where thehydrophilic polymer backbone helps maintain water solubility. TheOptiView DAB chromogen solutions were prepared with variable dextransulfate (9-29 kDa) polymer concentrations (0.25 to 5 wt %). Aprecipitate was observed with the OptiView DAB chromogen solutionscontaining less than 1 wt % dextran sulfate polymer (9-29 kDa). A higherDAB-to-polymer ratio occurred with lower wt % dextran sulfate polymersolutions, which increased the DAB-polymer complex hydrophobicity andcaused it to lose water solubility. No DAB sulfate precipitate wasobserved with the OptiView DAB chromogen solutions with greater then 2wt % dextran sulfate (9-29 kDa) upon addition of either 2.75 or 8.0 mMKHSO₄. A similar effect was observed with different molecular weightsized polystyrene sulfonate (PSS) polymers. Smaller PSS polymers, suchas 1.5 or 3.1 kDa PSS, were more prone to low DAB-polymer complexsolubility at lower wt % polymer concentrations than their largermolecular weight PSS polymers. Dextran sulfate polymers under 6 kDamolecular weight were also prone to lower DAB-polymer complexsolubility.

The influence of PSS polymer molecular weight on DAB IHC staining wasexamined using an iView detection method (tonsil tissue, Ki67). PSSpolymer stabilized DAB chromogen solutions were prepared using differentmolecular weight PSS at 2 wt %. Representative images are shown at 10×in FIGS. 3-8. FIG. 3 illustrates results from a tissue section stainedwith an OptiView DAB chromogen solution. FIG. 4-FIG. 8 illustratesresults from a tissue section stained with a 4 wt % polystyrenesulfonate (1.5 kDa, 5.1 kDa, 7.5 kDa, 16 kDa, and 35 kDa, respectively)stabilized DAB chromogen solutions. DAB IHC staining intensity andcoverage decreased with increasing PSS molecular weight. The netapparent DAB-PSS polymer complex molarity decreases with increasedpolymer molecular weight. A lower DAB concentration has been shown tocause the observed effects. The hematoxylin counterstain intensity andbackground increased with increased polymer molecular weight.Hematoxylin counterstain intensity and background are controlled bytissue pH during counterstain application. The DAB-PSS polymer complexdeposited on tissue will be deprotonated in Ventana Reaction Buffer andrequire further pH adjustment during counterstaining Polystyrenesulfonate maleic acid (PSSMA) co-polymers were not found under 15 kDamolecular weight to provide appropriate DAB IHC staining

Sulfate and sulfonate polymers do have some subtle impact on DAB tissuestaining; however, this impact can be largely controlled with polymersize and concentration. DAB tissue staining was reduced and hematoxylinbackground staining increased with larger polymers. Polymer wt %provided less impact on tissue staining with smaller polymers; however,optimization was required for polymer complex solubility and DAB sulfateprecipitate prevention. Top polymer candidates in this class were lowmolecular weight dextran sulfate (6-9.5 kDa) and polystyrene sulfonate(molecular weight=1.53 kDa, Mn=1.37 kDa). Representative images areshown in FIGS. 14-16. These figures illustrate results of a tissuesection stained with stabilized DAB chromogen solutions comprising nopolymer (FIG. 14); 4 wt % 1.53 kDa polystyrene sulfonate (FIG. 15); and4 wt % 6.5-10 kDa dextran sulfate (FIG. 16).

The sulfate and sulfonate polymer-stabilized DAB chromogen solutionswere thermally stressed at 45° C. to examine chromogen stability in thepresence of the new polymers. As previously stated, higher molecularweight PSS polymers are used in water purification for sequesteringpolyvalent metals. Polyvalent metal contaminants are known to catalyzeDAB oxidation. Sulfate and sulfonate polymers may contain potentialpolyvalent metal contaminants and result faster DAB oxidation rates.Dextran sulfates, PSS and PSSMA polymers have demonstrated the abilityto cause lower DAB chromogen stability (see Table 8). Dextran sulfateand PSSMA polymers were prone to forming DAB byproducts which wereobserved during HPLC analysis. These byproducts were not isolated orconfirmed by structural analysis. Polystyrene sulfonate (PSS) polymerscontained variable amounts of contaminants, which effect DAB oxidationrates. The PSS stabilized DAB chromogen solutions were as stable as thebase solution in certain cases.

TABLE 8 HPLC analytical data for polymer stabilized DAB chromogensolutions thermally stressed at 45° C. 1 d - 0° C. 5 d - 45° C. 25 d -45° C. DAB DAB DAB DAB base + 5 wt % Polymer 2HP % Δ 2HP % Δ 2HP % Δ NoPolymer Standard 4.60 n/a 4.57  −0.7 3.10 −33 9-20 kDa Dextran Sulfate4.15 −9.8 2.13 −53**  n/a†  −100** 6.5-10 kDa Dextran Sulfate 4.58 −0.43.92 −15** 0.45  −90** 1.53 kDa PSS 4.07 −12 3.70 −20  1.27 −72 5.18 kDaPSS 4.28 −7.0 4.10 −11  3.01 −35 15 kDa PSS Maleic Acid Co-polymer 4.04−12.2 2.31 −49** n/a n/a Low MW PSS Maleic Acid Co-polymer 4.15 −9.82.61 −43** n/a n/a **New HPLC byproduct peak(s) were present inchromogen solution. †No DAB remaining.

In summary, higher molecular weight polystyrene sulfonate (PSS) polymersare used in water purification for sequestering polyvalent metals.Polyvalent metal contaminants increase DAB oxidation rates. Dextransulfates, polystyrene sulfonates and polystyrene sulfonate maleic acid(PSSMA) polymers were leading polymers candidates in this group toreduce DAB sulfate precipitation. Polyvinylsufonic acid (4-6 kDa PVS, Nasalt) and polyethylene glycol sulfate (600 Da) did not form a watersoluble DAB-polymer complex. Small sulfate monomeric-like materials,namely 2-aminoethylhydrogen sulfate, did not form water soluble DABcomplexes.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for detecting a target in a tissue sample with aDAB chromogen or a derivative thereof, comprising: contacting the tissuesample with a specific binding moiety, the specific binding moiety beingspecific to the target, labeling the specific binding moiety with anenzyme conjugate, contacting the tissue sample with a composition forchromogenic immunohistochemistry, the composition comprising in asolution a solvent, a DAB chromogen or a derivative thereof, astabilizer, and a polyethyleneimine polymer soluble in the solvent,wherein the composition is configured to reduce precipitation of the DABchromogen or the derivative thereof under storage conditions butmaintain precipitation of the DAB chromogen or the derivative thereofunder detection conditions, contacting the tissue sample with anoxidant, wherein a reaction between the oxidant, the composition forchromogenic immunohistochemistry, and the enzyme conjugate causes theDAB chromogen or the derivative thereof to deposit proximally to thetarget, contacting the tissue sample with a counterstain, and detectingthe target in the tissue sample by locating the DAB chromogen or thederivative thereof.
 2. The method of claim 1, wherein labeling thespecific binding moiety includes detecting a hapten selected from anoxazole, a pyrazole, a thiazole, a nitroaryl compound other thandinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, arotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, or abenzodiazepine with an enzyme conjugate including an anti-haptenantibody specific to the hapten.
 3. The method of claim 2, wherein thehapten is selected from HQ, DIG, DNP, TS, NP, DCC, and biotin.
 4. Themethod of claim 1, wherein the polyethyleneimine polymer has an averagemolecular weight of between 1 kDa and about 500 kDa.
 5. The method ofclaim 1, wherein the polyethyleneimine polymer is about 0.05 percent toabout 10 percent of the composition for chromogenicimmunohistochemistry.
 6. The method of claim 1, wherein the enhancer isselected from the group consisting of imidazole, 2-hydroxypyridine andcombinations thereof.
 7. The methof of claim 1, wherein the stabilizeris sodium metabisulfate.
 8. The method of claim 1, wherein the enzyme isa peroxidase.
 9. The method of claim 8, wherein the peroxidase ishorseradish peroxidase
 10. The method of claim 1, wherein the tissuesample is a formalin-fixed, paraffin-embedded tissue sample.
 11. Themethod of claim 1, wherein storage conditions include a sealed storagecontainer not in fluid communication with other reagents and detectionconditions include the oxidant and the composition for chromogenicimmunohistochemistry deposited on the tissue sample and in contact withthe enzyme.
 12. The method of claim 1, wherein the specific bindingmoiety is an antibody or a nucleic acid probe.
 13. The method of claim1, wherein contacting the tissue sample with the composition forchromogenic immunohistochemistry comprises adding a first componentsolution comprising the DAB chromogen or the derivative thereof, asecond component solution comprising the stabilizer, and a thirdcomponent solution comprising the polymer to the tissue sample to formthe composition for chromogenic immunohistochemistry while in contactwith the tissue sample.
 14. A method of detecting a target in aformalin-fixed, paraffin-embedded tissue sample, comprising: contactingthe tissue sample with a haptinylated antibody, contacting the tissuesample with an enzyme conjugate comprising an anti-hapten antibody andhorseradish peroxidase, contacting the tissue sample with a chromogencomposition, the chromogen composition comprising a DAB chromogen, orderivative thereof, an enhancer selected from imidazole,2-hydroxypyridine, and combinations thereof; sodium metabisulfite; and apolymer selected from dextran sulfate, polystyrene sulfonate,polyethyleneimine, aminodextran, dextran DEAE, polystyrene sulfonatemaleic acid co-polymer, polyvinylsulfonate,poly(2-vinyl-1-methylpyridinium bromide,poly(2-methylacryloxethyltrimethylammonium bromide,poly(acrylamide/2-methylacryloxethyltrimethylammonium bromide, andcombinations thereof, contacting the tissue sample with hydrogenperoxidase, contacting the tissue sample with a hematoxylincounterstain, and detecting the target in the tissue sample.
 15. Amethod for detecting a target in a tissue sample with a DAB chromogen ora derivative thereof, comprising: contacting the tissue sample with aspecific binding moiety, the specific binding moiety being specific tothe target, labeling the specific binding moiety with an enzymeconjugate, contacting the tissue sample with a composition forchromogenic immunohistochemistry, the composition comprising in asolution a DAB chromogen, a stabilizer, and a polymer; wherein thestabilizer is sodium metabisulfite or sodium bisulfite, and the polymeris polyethyleneimine, wherein the composition is configured to remainstable under storage conditions so that the polymer complexes the DABchromogen or the derivative thereof so as to maintain the amount of theDAB chromogen or the derivative thereof in the solution, and wherein thecomposition is configured so that the DAB precipitates under detectionconditions to produce a signal suitable for chromogenicimmunohistochemistry. contacting the tissue sample with an oxidant,wherein a reaction between the oxidant, the composition for chromogenicimmunohistochemistry, and the enzyme conjugate causes the DAB chromogenor the derivative thereof to deposit proximally to the target,contacting the tissue sample with a counterstain, and detecting thetarget in the tissue sample by locating the DAB chromogen or thederivative thereof.
 16. The method of claim 15, wherein the DABchromogen is 3,3′-diaminobenzidine, and the stabilizer is sodiummetabisulfite.
 17. The method of claim 16, wherein the compositioncomprises about 1 to about 15 mM 3,3′-diaminobenzidine, about 0.1 toabout 6 mM sodium metabisulfite and about 0.05% to about 0.5% (w/w)polyethyleneimine.